Listeria monocytogenes (L.m) is an intracellular bacterial pathogen that causes serious foodborne illness in pregnant women, the elderly and immunocompromised individuals. Listeriolysin O (LLO), a pore-forming cytolysin, and two bacterial phospholipases, PI-PLC and PC-PLC, are essential determinants of pathogenesis that mediate lysis of host cell vacuoles resulting from bacterial entry and intracellular spread. LLO is also expressed during intracytosolic growth and mediates numerous alterations in host cell physiology. During in vitro infection of cell lines, LLO is sufficient to facilitate lysis of all vacuolar membranes. Yet, PI-PLC and PC-PLC increase the efficiency of membrane lysis. It is hypothesized that these determinants play a specific role in the dissolution of each vacuolar membrane and the intracytosolic production of LLO is necessary for optimal intracellular growth. The focus of this proposal is to precisely define the temporal requirement of LLO for intracellular growth and cell-to-cell spread in primary host cells and for the maintenance of in vivo infection in a mouse infection model. In Aim I, the precise requirement for LLO expression during intracellular growth and spread in primary host cells will be determined. This will be accomplished by using a novel genetic approach to allow regulated production of LLO during intracellular infection. Intracellular LLO levels will be varied during infection of primary host cells and bacterial replication and spread determined by microscopic analysis and enumeration of intracellular bacteria. In Aim II, the precise roles of LLO, PI-PLC and PC-PLC in dissolution of vacuolar membranes during intracellular spread will be identified. L.m strains allowing regulated expression of LLO in PI-PLC and PC-PLC mutants will be used in mixed host cell infections. Plaque formation in cell monolayers, differential time-lapse fluorescence microscopy and high-resolution electron microscopy will be used to evaluate progression of infection and dissolution of vacuolar membranes. In Aim III, we will evaluate the requirement of LLO expression for maintenance of in vivo infection and the development of acquired immunity. BALB/c mice will be infected under varying times of in vivo LLO induction. Progression of infection will be evaluated by enumerating bacteria from organs and comparing to infection of wild-type and defined L.m mutants. Immunological assessment will be determined by ELISPOT analysis and protection from wild-type bacterial challenge.